Marco Pezzullo

Association between Serum Atypical Fibroblast Growth Factors 21 and 19 and Pediatric Nonalcoholic Fatty Liver Disease.

Atypical fibroblast growth factors (FGF) 21 and 19 play a central role in energy metabolism through the mediation of Klotho coreceptor. Contradictory findings are available about the association of FGF21 and FGF19 with nonalcoholic fatty liver disease (NAFLD) in humans. We investigated the association of serum FGF21, FGF19 and liver Klotho coreceptor with non-alcoholic steatohepatitis (NASH) and fibrosis in children with NAFLD. Serum FGF21 and FGF19 were measured in 84 children with biopsy-proven NAFLD and 23 controls (CTRL). The hepatic expression of Klotho coreceptor was measured in 7 CTRL, 9 patients with NASH (NASH+) and 11 patients without NASH (NASH−). FGF21 and FGF19 showed a tendency to decrease from CTRL (median FGF21 = 196 pg/mL; median FGF19 = 201 pg/mL) to NASH− (FGF21 = 89 pg/mL; FGF19 = 81 pg/mL) to NASH+ patients (FGF21 = 54 pg/mL; FGF19 = 41 pg/mL) (p<0.001 for all comparisons) and were inversely associated with the probability of NASH and fibrosis in children with NAFLD. The hepatic expression of Klotho coreceptor was inversely associated with NASH (R2 = 0.87, p<0.0001) and directly associated with serum FGF21 (R2 = 0.57, p<0.0001) and FGF19 (R2 = 0.67, p<0.0001). In conclusion, serum FGF19 and FGF21 and hepatic Klotho expression are inversely associated with hepatic damage in children with NAFLD and these findings may have important implications for understanding the mechanisms of NAFLD progression.

Deregulation of the IL-1β axis in chronic recurrent multifocal osteomyelitis.

BackgroundThis study aims to investigate the inflammasome response in peripheral blood mononuclear cells (PBMCs) and the expression of inflammasome components in bone biopsies from patients with chronic recurrent multifocal osteomyelitis (CRMO).MethodsThe expression of inflammasome components mRNAs was evaluated in PBMCs isolated from 15 CRMO patients and 13 healthy controls by quantitative real-time PCR. The Interleukin (IL)-1β released in the medium of PBMC cultures after treatment with lipopolysaccharides (LPS) alone or LPS and ATP was measured by ELISA. Immunohistochemical staining for Apoptosis-associated Speck-like protein (ASC), caspase-1 (CASP-1), Nod-like receptor protein-3 (NLRP3) and IL-1β expression was performed in bone biopsies from CRMO patients.ResultsmRNA levels of ASC, CASP-1 and IL-1β were significantly higher in freshly isolated PBMCs from CRMO patients in active disease than in healthy controls. CASP-1 and IL-1β transcript levels were significantly higher also in PBMCs from CRMO patients in remission compared to healthy controls. PBMCs from CRMO patients in active disease stimulated in vitro with LPS showed a significant increase in IL-1β release compared to healthy control cells. Immunohistochemistry staining of bone tissue revealed the expression of inflammasome components in CRMO osteoclasts.ConclusionsOur data suggest that an abnormal regulation of IL-1β axis may be involved in CRMO pathogenesis.

IRF1 and NF-kB Restore MHC Class I-Restricted Tumor Antigen Processing and Presentation to Cytotoxic T Cells in Aggressive Neuroblastoma

Neuroblastoma (NB), the most common solid extracranial cancer of childhood, displays a remarkable low expression of Major Histocompatibility Complex class I (MHC-I) and Antigen Processing Machinery (APM) molecules, including Endoplasmic Reticulum (ER) Aminopeptidases, and poorly presents tumor antigens to Cytotoxic T Lymphocytes (CTL). We have previously shown that this is due to low expression of the transcription factor NF-kB p65. Herein, we show that not only NF-kB p65, but also the Interferon Regulatory Factor 1 (IRF1) and certain APM components are low in a subset of NB cell lines with aggressive features. Whereas single transfection with either IRF1, or NF-kB p65 is ineffective, co-transfection results in strong synergy and substantial reversion of the MHC-I/APM-low phenotype in all NB cell lines tested. Accordingly, linked immunohistochemistry expression patterns between nuclear IRF1 and p65 on the one hand, and MHC-I on the other hand, were observed in vivo. Absence and presence of the three molecules neatly segregated between high-grade and low-grade NB, respectively. Finally, APM reconstitution by double IRF1/p65 transfection rendered a NB cell line susceptible to killing by anti MAGE-A3 CTLs, lytic efficiency comparable to those seen upon IFN-γ treatment. This is the first demonstration that a complex immune escape phenotype can be rescued by reconstitution of a limited number of master regulatory genes. These findings provide molecular insight into defective MHC-I expression in NB cells and provide the rational for T cell-based immunotherapy in NB variants refractory to conventional therapy.

Association between mannose-binding lectin gene polymorphisms and necrotizing enterocolitis in preterm infants.

Objectives: The aim of the present study was to evaluate whether polymorphisms of the mannose-binding lectin (MBL-2) gene and MBL serum levels on admission to neonatal intensive care unit are associated with necrotizing enterocolitis (NEC) in preterm infants and to verify MBL expression in NEC bowels. Methods: In this retrospective cohort study, 107 neonates (41 with NEC and 66 controls) were included. MBL-2 genotyping for the promoter polymorphism −221 and for the exon 1 variant alleles at codons 52, 54, and 57 was performed. MBL levels were determined by enzyme-linked immunosorbent assay in 55 infants. Immunohistochemical staining for MBL expression was performed on bowel specimens. The main study outcome was severe NEC (Bell stages II/III). Results: The −221 Y allele and the MBL-2 YY genotype were more frequent in neonates with severe NEC than in controls (P = 0.04 and P = 0.004, respectively). In the multivariate analysis, the MBL-2 YA/YA genotype was associated with NEC (odds ratio = 3.03, 95% confidence interval 1.13%–8.13%, P = 0.024). Neonates with NEC had MBL level on admission >400 ng/mL more frequently than controls (P = 0.043). Among neonates with severe NEC, the deceased neonates were carriers of high or intermediate producing MBL-2 genotypes (P = 0.035). Finally, MBL was highly expressed in intestinal tissue from infants with NEC. Conclusions: MBL-2 genotypes associated with high MBL serum levels represent a risk factor for NEC. This finding, together with the MBL expression in bowel tissue, supports a role for MBL in the pathogenesis of NEC.

Src nuclear localization and its prognostic relevance in human osteosarcoma

Osteosarcoma is the most common malignant bone tumor in children and young adults. The identification of proteins which exhibit different subcellular localization in low‐ versus high‐risk osteosarcoma can be instrumental to obtain prognostic information and to develop innovative therapeutic strategies. Beside the well‐characterized membrane and cytoplasmic localization of Src protein, this study evaluated the prognostic relevance of its so‐far unknown nuclear compartmentalization. We analyzed the subcellular distribution of total and activated (pY418) Src in a tissue microarray including 60 osteosarcoma samples. Immunohistochemical analyses revealed a variable pattern of Src expression and localization, ranging from negative to high‐stained nuclei combined with a substantial cytoplasmic staining for total and activated forms. The analysis of Kaplan–Meier survival curves in relationship to the diverse permutations of cytoplasmic and nuclear staining suggested a correlation between Src subcellular localization and the overall survival (OS) of osteosarcoma patients. In order to explain this different subcellular localization, normal osteoblasts and three osteosarcoma cell lines were used to investigate the molecular mechanism. Once confirmed a variable Src localization also in these cell lines, we demonstrated a correlation between the N‐myristoyltransferase enzymes expression and activity and the Src nuclear content. In conclusion, these results described a so‐far unknown Src nuclear localization in osteosarcoma cells, suggesting that the combined detection of nuclear and cytoplasmic Src levels can be used as a prognostic marker for osteosarcoma patient survival. A correlation between the N‐myristoyltransferase enzymes and the Src subcellular localization was described as well.

Evaluation of Endoglin (CD105) expression in pediatric rhabdomyosarcoma

BackgroundThe Intratumoral Microvessel Density (IMVD) is commonly used to quantify tumoral vascularization and is usually assessed by pan-endothelial markers, such as CD31. Endoglin (CD105) is a protein predominantly expressed in proliferating endothelium and the IMVD determined by this marker measures specifically the neovascularization. In this study, we investigated the CD105 expression in pediatric rhabdomyosarcoma and assessed the neovascularization by using the angiogenic ratio IMVD-CD105 to IMVD-CD31.MethodsParaffin-embedded archival tumor specimens were selected from 65 pediatric patients affected by rhabdomyosarcoma. The expression levels of CD105, CD31 and Vascular Endothelial Growth Factor (VEGF) were investigated in 30 cases (18 embryonal and 12 alveolar) available for this study. The IMVD-CD105 to IMVD-CD31 expression ratio was correlated with clinical and pathologic features of these patients.ResultsWe found a specific expression of endoglin (CD105) in endothelial cells of all the rhabdomyosarcoma specimens analyzed. We observed a significant positive correlation between the IMVD individually measured by CD105 and CD31. The CD105/CD31 expression ratio was significantly higher in patients with lower survival and embryonal histology. Indeed, patients with a CD105/CD31 expression ratio < 1.3 had a significantly increased OS (88%, 95%CI, 60%–97%) compared to patients with higher values (40%, 95%CI, 12%–67%). We did not find any statistical correlation among VEGF and EFS, OS and CD105/CD31 expression ratio.ConclusionCD105 is expressed on endothelial cells of rhabdomyosarcoma and represent a useful tool to quantify neovascularization in this tumor. If confirmed by further studies, these results will indicate that CD105 is a potential target for combined therapies in rhabdomyosarcoma.

Biophysical and biological contributions of polyamine-coated carbon nanotubes and bidimensional buckypapers in the delivery of miRNAs to human cells

Recent findings in nanomedicine have revealed that carbon nanotubes (CNTs) can be used as potential drug carriers, therapeutic agents and diagnostics tools. Moreover, due to their ability to cross cellular membranes, their nanosize dimension, high surface area and relatively good biocompatibility, CNTs have also been employed as a novel gene delivery vector system. In our previous work, we functionalized CNTs with two polyamine polymers, polyethyleneimine (PEI) and polyamidoamine dendrimer (PAMAM). These compounds have low cytotoxicity, ability to conjugate microRNAs (such as miR-503) and, at the same time, transfect efficiently endothelial cells. The parameters contributing to the good efficiency of transfection that we observed were not investigated in detail. In fact, the diameter and length of CNTs are important parameters to be taken into account when evaluating the effects on drug delivery efficiency. In order to investigate the biophysical and biological contributions of polymer-coated CNTs in delivery of miRNAs to human cells, we decided to investigate three different preparations, characterized by different dimensions and aspect ratios. In particular, we took into account very small CNTs, a suspension of CNTs starting from the commercial product and a 2D material based on CNTs (ie, buckypapers [BPs]) to examine the transfection efficiency of a rigid scaffold. In conclusion, we extensively investigated the biophysical and biological contributions of polyamine-coated CNTs and bidimensional BPs in the delivery of miRNAs to human cells, in order to optimize the transfection efficiency of these compounds to be employed as efficient drug delivery vectors in biomedical applications.

Abstract 3417: Ezh2 is up-regulated and correlates with Ki67 and CD31 expression in human pediatric rhabdomyosarcoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Rhabdomyosarcoma (RMS) is a pediatric tumor believed to originate from perturbations of the normal development of the muscle lineage. The Polycomb Group (PcG) protein Ezh2, which was originally implicated in inhibiting homeobox gene expression during embryonic development, was recently shown to act as a negative regulator of muscle differentiation. Moreover, Ezh2 is a histone methyltransferase that is aberrantly overexpressed in most human cancers. Comparing the level of the expression of Ezh2 in RMS cell lines, a small group of tumor samples with normal skeletal muscle myoblasts (SkMC), and normal muscle tissues, we have previously observed that Ezh2 is highly upregulated in rhabdomyosarcoma tumors (Ciarapica et al., 2009). The aim of this study was to characterize the relevance of Ezh2 expression in RMS. Ezh2 expression was analyzed by immunohistochemistry in a cohort of 32 RMS primary tumors and correlated with the expression of the cell proliferation marker, Ki67, and the angiogenic marker, CD31. Results of these expression studies were then correlated with the clinicopathological features of the tumor samples. No significant difference in the clinicopathological variables was observed between the two major histophatological groups of RMS, the most aggressive alveolar RMS (ARMS) and the embryonal RMS (ERMS). Ezh2 expression was not correlated with the clinical parameters of the cohorts, but strikingly, a significant positive correlation was observed between Ezh2 and Ki67 expression in the whole RMS sample (r0 = 0,560; p = 0,013). The number of the large vessels was correlated with the total number of vessels (r0= 0,730; p<0,0001) and with the expression of Ki67 (r0= 0,421; p=0,02). In ARMS samples, the number of large vessels was significantly correlated with the expression of Ezh2 (r0= 0,559; p=0,047), the total number of vessels (r0= 0,781; p=0,02), and the expression of Ki67 (r0= 0,738; p=0,004). This study confirmed our previous observation regarding the aberrantly high expression of Ezh2 in rhabdomyosarcoma tumors on a larger cohort of samples. Moreover, the present data established a positive correlation between Ezh2 expression and the proliferative and angiogenic potential of rhabdomyosarcoma neoplasia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3417.